The overall goal of this proposal is to elucidate the mechanisms associated with decreased cerebral artery diameter (vasospasm) following subarachnoid hemorrhage (SAH). Vasospasm is the major cause of death and disability in aneurysmal SAE patients. Despite the devastating consequences of SAE-induced vasospasm, only modest progress has been made in defining the cellular events leading to this disorder. From the intact animal to the molecular level, this proposal takes an integrated approach to expand current knowledge regarding the mechanisms underlying SAH-induced vasospasm. Global intracellular Ca2+ plays a central role in cerebral artery contractility and has also been shown to influence gene expression. Our proposed work will apply innovative techniques to study alterations in Ca Z+ handling and Ca2+-dependent changes in gene transcription that may contribute to SAH-induced vasospasm. Our integrative approach includes acute studies in isolated cerebral arteries, a rabbit model of SAE, and prolonged in vitro exposure of cerebral arteries to oxyhemoglobin. Specific Aim 1 applies patch clamp electrophysiology, calcium imaging and biochemical techniques to examine the molecular mechanisms associated with oxyhemoglobin-induced cerebral artery constriction. This Specific Aim will build upon our preliminary data to examine the role of plasma membrane ion channels, sarcoplasmic reticulum Ca 2+ stores, and the small GTPase RhoA in this phenomenon. Specific Aim 2 is to understand alterations in Ca 2+ handling associated with cerebral artery vasospasm using a rabbit model of SAE. This animal model provides the exciting opportunity to combine a broad spectrum of techniques to examine cellular events linking SAE to cerebral artery vasospasm. In Specific Aim 3, organ culture techniques will be used to assess the long-term impact of oxyhemoglobin on intact cerebral arteries. This information regarding the selective long-term application of oxyhemoglobin should greatly expand current knowledge regarding the role of this agent in SAH-induced vasospasm. Additional benefits from this Specific Aim will be the application of organ culture techniques to extend the study period for human cerebral artery segments and the development of an in vitro SAE vasospasm model that should substantially reduce animal use. In summary, this study should enhance current knowledge of the mechanisms underlying SAH-induced vasospasm and may provide insight into novel therapeutic approaches for the treatment of this disease.